Project Summary There are fundamental gaps in understanding how alcoholism leads to alcohol withdrawal syndrome, with its range of serious clinical symptoms, including profound seizures. Withdrawal seizures become progressively worse with each withdrawal, complicate treatment, and can be fatal. It is therefore vital to identify safe and effective treatment alternatives. Calcium channels may offer such an alternative for treatment. The long-range goal of this work is to better understand the cellular and molecular mechanisms underlying alcohol withdrawal. The immediate goals of this proposal are: 1) to define the cellular and molecular alterations in T-type Ca2+ channels that occur in response to multiple WDs~ 2) to link these changes to the development of seizure and other features of alcohol WD syndrome~ 3) to examine whether treatments targeting these channels are effective~ 4) to understand how the acute sensitivity of the T-channel isoform implicated in our studies relates to alcohol WD syndrome and seizures involving this same channel~ and 5) to determine how relevant brain circuitry is recruited into WD seizure. Guided by strong preliminary data and published findings, and enabled by an innovative method of studying WD seizures, the following specific aims will be pursued: Aim 1 will determine how acute inhibition of T-type channels by ethanol leads to WD hyperexcitability. Using the finding that PKC mediates the acute inhibitory effect of ethanol, whole cell recordings will be used to test the hypothesis that PKC inhibitors will reveal a change in ethanol-mediated inhibition of T-type currents during WD. Aim 2 will determine the contribution of T-type channels to seizures resulting from multiple ethanol withdrawals. Quantitative EEG recordings will be used to test whether T-type channel blockers will inhibit WD seizure and will protect against the progression of seizures due to multiple, intermittent WDs. Aim 3 will determine the site of origin and propagation patterns of WD seizure. The progression of WD seizure will be characterized using in vivo, multisite recordings coupled with novel optogenetic activation of this network using channelrhodopsin 2. The studies are highly significant because of the vertically integrated investigation of the role of T-type calcium channels in alcohol withdrawal seizure, which is an enduring problem in the treatment of abstaining alcoholics. The principles of channel modulation and network involvement promise to extend to other neural systems that include these channels. The studies are innovative because they deliver a new optogenetic approach to studying WD seizures, which will be used to understand the progression of WD seizure in unprecedented detail, and a potential new therapy based on a current antiepileptic drug.